This grant application is designed to study the mechanism of gonadotropin interaction with testicular receptors as related to the concepts of testicular activation and desensitization. Our primary objective is to determine whether gonadotropin binding is related to "receptor occupancy" or to its "loss" via dissociation of loosely coupled binding component from adenylate cyclase at the membrane level. According to our working hypothesis the binding component and the adenylate cyclase constitute the physiologic receptors and we envision "receptor dissociation" as one of the mechanisms associated with the activation of adenylate cyclase which in turn leads to the adenylate cyclase inactivation when the process of receptor dissociation from the broken cell preparations is completed. To study this phenomenon in greater detail and to further differentiate between the processes of receptor dissociation - biologic response, receptor loss - desensitization and receptor synthesis - renewal of tissue sensitivity in intact tissue and Leydig cells, we propose to investigate the mechanism by which hormone activates and desensitizes the cell. Experiments involving incubation of dispersed Leydig cells with hormones, ethanol and other water miscible organic solvents are proposed to study the specificity of ethanol action. Six parameters to be determined are gonadotropin receptor sites, the adenylate cyclase activity, serum gonadotropins, total cAMP contents, steriodogenic output, and ethanol-soluble factor activities. The integration of these results, along with those of in vitro studies to replenish gonadotropin receptors following their complete depletion under optimal conditions by agents such as prolactin and other hormones through perifusion technique, should provide the first direct evidence of de novo receptor synthesis and its translocation at the membrane level. In perifusion experiments all six parameters will be measured to establish the criteria for receptor synthesis, translocation, and dissociation. Furthermore, the binding model that we have already proposed will be supported by studies on the cAMP-cAMP protein kinase system. We shall attempt to purify ethanol-soluble factors (i.e. factors) in order to convert inactive adenylate cyclase into an active cyclase, and we shall attempt to characterize the soluble factors as a prerequisite to the study of their biosynthesis.